Abstract
To gain insight into the function and organization of proteins assembled on the DNA in response to genotoxic insult we investigated the phosphorylation of the Schizosaccharomyces pombe PCNA-like checkpoint protein Rad9. C-terminal T412/S423 phosphorylation of Rad9 by Rad3(ATR) occurs in S phase without replication stress. Rad3(ATR) and Tel1(ATM) phosphorylate these same residues, plus additional ones, in response to DNA damage. In S phase and after damage, only Rad9 phosphorylated on T412/S423, but not unphosphorylated Rad9, associates with a two-BRCT-domain region of the essential Rad4(TOPBP1) protein. Rad9-Rad4(TOPBP1) interaction is required to activate the Chk1 damage checkpoint but not the Cds1 replication checkpoint. When the Rad9-T412/S423 are phosphorylated, Rad4(TOPBP1) coprecipitates with Rad3(ATR), suggesting that phosphorylation coordinates formation of an active checkpoint complex.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Binding Sites / genetics
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Cell Cycle Proteins / chemistry
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Cell Cycle Proteins / genetics
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Cell Cycle Proteins / metabolism*
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Checkpoint Kinase 1
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Checkpoint Kinase 2
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DNA Damage
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DNA Replication
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DNA, Fungal / genetics
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DNA, Fungal / metabolism
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DNA-Binding Proteins / metabolism*
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Enzyme Activation
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Models, Biological
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Mutation
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Phosphorylation
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Protein Kinases / chemistry
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Protein Kinases / genetics
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Protein Kinases / metabolism*
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S Phase
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Schizosaccharomyces / cytology
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Schizosaccharomyces / genetics
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Schizosaccharomyces / metabolism
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Schizosaccharomyces pombe Proteins / chemistry
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Schizosaccharomyces pombe Proteins / genetics
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Schizosaccharomyces pombe Proteins / metabolism*
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Transglutaminases / metabolism*
Substances
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Cell Cycle Proteins
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DNA, Fungal
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DNA-Binding Proteins
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RAD4 protein, S pombe
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Schizosaccharomyces pombe Proteins
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rad9 protein
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Transglutaminases
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Protein Kinases
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Checkpoint Kinase 2
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Checkpoint Kinase 1
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Chk1 protein, S pombe
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rad3 protein, S pombe